Caco 2

The human epithelial cell line Caco2 (carcinoma colon-2 cell, American Type Culture Collection, Manassas, VA) is not only accepted as a model for intestinal barrier [36] (link) but for fructose metabolism [37] (link) as well. Caco2 cells, passages 37–47, were maintained in Dulbecco's modified Eagles' medium high glucose (4,5 g/l, PAA Laboratories, Pasching, Austria) supplemented with 20% fetal bovine serum (FBS) (Biochrom AG, Berlin Germany), 1% non-essential amino acids (Biochrom AG, Berlin Germany) and 1% penicillin-streptomycin (PAA Laboratories, Pasching, Austria) in a humidified 5% CO₂ atmosphere at 37°. Cells were fed every 2 or 3 days and transferred after reaching 75% of confluence to transwell systems (transparent PET Membrane, 0.4 µm pore size, 4.2 cm² growth area; BD Falcon, Heidelberg, Germany) at a density of 5*10⁵ cells per well.
After 13 days cells differentiated completely and were maintained with DMEM supplemented with 1% NEA and 1% penicillin-streptomycin, without FBS 24 h.

The human colon adenocarcinoma cell line Caco-2 (ECACC, Porton Down, UK) was cultured in Dulbecco's modified Eagle's medium (DMEM; PAA Laboratories GmbH, Coelbe, Germany) supplemented with 10 % fetal calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a humidified atmosphere of 5 % CO₂. Cells from passages 29-40 were used for all experiments. Caco-2 cells were cultured in six-well plates until complete confluency and were further cultivated for 14 to 18 days in order to allow differentiation. For a recent detailed description of Caco-2 cell culture, please refer to Lichtenstein et al. (2017[19 ]). Verification of successful differentiation into enterocyte-like cells, which was also used as a verification of cell line identity for Caco-2 cells, has also been described in detail in the aforementioned previous publication (Lichtenstein et al., 2017[19 ]).

The CRC cell lines Caco-2 (KRAS wild-type) and HRT-18 (KRAS-mutated) were purchased from American Type Culture Collection. Caco-2 was cultured in MEM with Earle’s salts (PAA Laboratories, Pasching, Austria) supplemented with 2 mmol/L of l-glutamine, 1.5 g/L of sodium bicarbonate, 0.1 mmol/L of nonessential amino acids, 50 units/mL of penicillin, 50 µg/mL of streptomycin, and 10% FBS (PAA Laboratories). HRT-18 cells were maintained in RPMI 1640 (GIBCO, Vienna, Austria) containing 2 mmol/L of l-glutamine, 50 units/mL of penicillin, 50 µg/mL of streptomycin, and 10% FBS. After obtaining a confluence of approximately 80%, total RNA was isolated following a standard TRIzol protocol and RNA was stored at −70 °C until further procedures.